Method of diagnosis and kit of parts therefor

ABSTRACT

A method for diagnosing coeliac disease in a patient comprising (a) providing an antibody sample from the patient to be tested, (b) contacting said antibody sample with cross-linked extracellular matrix (ECM) material immobilized on a surface and (c) detecting binding of antibodies present in the sample to the ECM-coated surface, wherein the binding of antibodies present in the sample to the ECM-coated surface being indicative of a positive diagnosis. Preferably, step (c) comprises an ELISA. The invention further provides a kit of parts for use in diagnosing coeliac disease in a patient.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a National Phase application under 35 U.S.C. § 371of International Application No. PCT/GB2002/03276 filed Jul. 18, 2002and claims the benefit of Great Britain Application No. 0117870.6 filedJul. 21, 2001.

The present invention relates to a method of diagnosing coeliac disease.The invention further provides a kit of parts for the diagnosis ofcoeliac disease.

BACKGROUND

Coeliac disease (CD) is a complex immunological response to the presenceof wheat gluten in the gut. Generally the disease manifests throughmalabsorption of the upper small intestine caused by villus atrophy andcrypt hyperplasia resulting from the inappropriate immune response togluten in this region. Symptoms of the disease often reflect themalabsorption occurring in the gut, i.e. growth defects, anaemia anddiarrhoea although asymptomatic patients are common. If left untreatedthe disease may lead to complications such as small intestinal lymphoma(Logan et al., 1989, Gastroenterology 97:265-271), and small boweladenocarcinoma (Begos et al., 1995, J Clin Gastroenterol 20:233-6).

Until recently the recognised method of diagnosis for coeliac disease asdefined by the European Society for Paeditric Gastroenterology andNutrition (ESPGAN) was a source of considerable stress to patients dueto repeated intestinal biopsy over long periods of time. Extensiveresearch into the progression of the disease and into non-invasive toolsfor its diagnosis has led to the identification of several broadlydefined serological markers. In the light of these discoveries EPSGANhave revised their criteria for CD diagnosis (Walker-Smith et al, 1990,Report of working group of European Society of PediatricGastroenterology and Nutrition) so that biopsy is limited to one sample,taken at presentation, along with the presence of recognised CDserological markers that diminish with gluten withdrawal from the diet.Thus the research emphasis has shifted in recent years to the search fora serological test that is as at least as good as gut biopsy for CDdiagnosis.

To date, the humoral aspect of coeliac disease has been groupedaccording to the antigens with which reaction has been shown in in vitroassays. Thus both IgG and IgA class anti-gliadin antibodies (AGA),anti-reticulin antibodies (ARA), and anti-endomysial antibodies (EMA)have been defined and investigated. Many papers, reviews and trials havebeen published on the relative efficacy of using each of these markersin various assay formats and each approach has its champion. Doubts overthe specificity of AGA have persisted as it has become apparent thatlimited immune responses to gliadin along with other recognised foodantigens can occur in a variety of gut disorders (Maki et al, 1995, InHowdle P D (ed): Coeliac Disease, Baillières Clin Gastroenterol9:231-249). Of the other two groups of antibody the EMA have risen toprominence with the discovery by Dieterich et al. (1997, Nature Med3:797-801) that Type II transglutaminase could be precipitated from cellextracts using AGA depleted serum from patients with chronic CD. Thehuman Type II TG based ELISAs that rapidly followed proved to be bothhighly specific and sensitive and were obviously widely welcomed as areplacement for the antiquated and difficult to interpretimmunofluorescent test using monkey oesophagus or human placentasections.

It was soon apparent however that the results of clinical use of type IITG based assays very often failed to match these high levels ofreliability. This inconsistency in the results may reflect the fact thatmuch of the original research has been performed using sera from chroniccoeliac patients whereas the clinical use of the assay has seen a widervariety of patients that have presented the disease in differing degreesof severity.

In addition to the poor clinical results seen with type II TG basedELISAs it is now becoming clear that several other antigens are involvedin the autoimmune aspect of coeliac disease (Clemente et al., 2000, Gut47:520-526) and that the original inference of Dieterich that type II TGis the only significant antigen has become invalid.

Hence, there is a need to improved methods of diagnosing coeliacdisease.

SUMMARY OF THE INVENTION

Accordingly, a first aspect of the invention provides method fordiagnosing coeliac disease in a patient comprising:

-   -   (a) providing an antibody sample from the patient to be tested;    -   (b) contacting said antibody sample with cross-linked        extracellular matrix (ECM) material immobilised on a surface;        and    -   (c) detecting binding of antibodies present in the sample to the        ECM-coated surface        wherein the binding of antibodies present in the sample to the        ECM-coated surface is indicated of a positive diagnosis.

Preferably, the antibody sample is a serum sample. Alternatively, theantibody sample may be a saliva sample.

Conveniently, the antibody sample comprises IgA antibodies.

By ‘cross-linked extracellular matrix (ECM) material’ we mean proteinssecreted by cells which are deposited in an insoluble matrix surroundingthe cells or are specific to a particular cell membrane. Proteinsnormally associated with the ECM include fibronectin, vitronectin,collagens, latent TGFβ-1 binding protein (LTBP-1), fibrillins, elastinand laminin. (Comper, W D, 1996, Extracellular matrix, Vol I and II,Harwood Acad. Pubs, Amsterdam, Netherlands).

Preferably, the ECM is cross-linked by the action of a tissuetransglutaminase enzyme, which catalyzes the formation of inter- andintra-protein ε(γ-glutamyl) isopeptide bonds and the incorporation ofpolyamines at certain glutamine residues (Greenberg, C. S.,Birckbichler, P. J. and Rice, R. H. (1991) FASEB J. 5, 3071-3077).

Conveniently, the ECM material is adhered to the surface.

Preferably, ECM is derived from cells which express a tissuetransglutaminase. Advantageously, the ECM is derived from mammaliancells, for example human or murine cells.

Thus, the method comprises measuring antibodies in a sample from thepatient to be tested which have affinity for antigenic components of thecross-linked ECM material. For example, the antibodies may have affinityfor extracellular matrix proteins, tissue transglutaminase or novelproducts of the cross-linking activity of the transglutaminase on theextracellular matrix proteins.

Advantageously, step (c) comprises simultaneously detecting binding oftwo or more types of antibody, each type of antibody having affinity fora different component of the cross-linked ECM.

Preferably, step (c) comprises detecting binding of IgA antibodies tothe ECM-coated surface.

Alternatively, step (c) comprises detecting binding of IgG antibodies tothe ECM-coated surface.

In a preferred embodiment of the first aspect of the invention, adiagnosis is obtained by comparing the level of binding of antibodies inthe sample from the patient to the level of binding of antibodies in anantibody sample from a donor who does not have coeliac disease. Thus,the method may further comprise repeating steps (b) and (c) with anegative control sample. Conveniently, the negative control sample is aserum sample or a saliva sample from a normal healthy individual.

Preferably, a positive diagnosis is made when the level of binding ofantibodies present in the patient's sample to the ECM-coated surface isat least 20%, 30%, 40%, 50%, 100%, 200% or more greater than the levelof binding of antibodies present in the negative control sample. Morepreferably, a positive diagnosis is made when the level of binding ofantibodies present in the patient's sample to the ECM-coated surface isat least 100% greater than the level of binding of antibodies present inthe negative control sample.

In a preferred embodiment of the first aspect of the invention, step (c)comprises using an enzyme-linked immunosorbent assay (ELISA). Suchmethodology is well-known in the art (for example, see Sblattero et al,2000, Am J. Gastroenterol. 95:1253-57).

In conventional (indirect) ELISAs, the binding of a (primary) antibodyto a target antigen is detected by means of a secondary antibody withaffinity for the primary antibody. The secondary antibody is conjugatedto an enzyme, such as horse radish peroxidase, which catalyses thetransformation of a non-detectable substrate to a detectable product.Thus, the detectable product gives a measure of the binding of theprimary antibody. Often, the detectable product is coloured and may bedetected by spectrophotometry.

In the methods of the invention, the target molecules are anti-ECMantibodies present in the antibody sample from the patient to be tested,hence direct ELISAs may be used in which an anti-human Ig antibodyconjugated to a enzyme such as horse-radish peroxidase (HRP) is applieddirectly to the sample to be tested.

Advantageously, the ELISA comprises using an anti-human IgA antibodyconjugated to HRP to detect binding of antibodies in sera or salivasamples to the cross-linked ECM.

A second aspect of the invention provides a kit of parts for diagnosingcoeliac disease for use in the method of the invention, the kitcomprising

-   -   (a) a surface upon which cross-linked extracellular matrix (ECM)        material is immobilised or means for producing such as surface;        and    -   (b) means for detecting binding to said ECM material of        antibodies produced by persons suffering from coeliac disease.

Preferably, the kit comprises a surface upon which cross-linked ECMmaterial is immobilised. Advantageously, the ECM material is adhered tothe surface. Conveniently, the cross-linked ECM material is derived fromcells which secrete and deposit ECM and which express a tissuetransglutaminase. For example, the cells may be human foreskin dermalfibroblasts. Suitably, the cells naturally express a tissuetransglutaminase at high levels.

In an alternative embodiment, the cross-linked ECM material is derivedfrom cells capable of secreting and depositing ECM which naturallyexpress low levels of a tissue transglutaminase (or which do notnaturally express a tissue transglutaminase) but are transfected with anucleic acid molecule encoding a transglutaminase so that expression ofthe enzyme is increased. Preferably, the cells are fibroblast cells,such as Swiss 3T3 fibroblast cells, transfected with a nucleic acidmolecule encoding tissue transglutaminase, as described in Verderio etal. (1998) Exp. Cell Res. 239:119-38.

By ‘a transglutaminase’ we mean a member of the group of enzymesidentified by Enzyme Commission System of Classification No. 2.3.2.13(EC 2.3.2.13). Preferably, the transglutaminase is human type IItransglutaminase.

Exemplary nucleotide sequences encoding tissue transglutaminase areknown in the art. For example, the coding sequence for human tissuetransglutaminase is disclosed in Gentile et al, 1991, J. Biol. Chem.266(1) 478-483 (Accession no. M55153).

Nucleic acid molecules encoding a transglutaminase may be used inaccordance with known techniques, appropriately modified in view of theteachings contained herein, to construct an expression vector, which isthen used to transform an appropriate host cell for the expression andproduction of the polypeptide of the invention. Methods of expressingproteins in recombinant cells lines are widely known in the art (forexample, see Sambrook & Russell, 2001, Molecular Cloning, A LaboratoryManual, Third Edition, Cold Spring Harbor, N.Y.). Exemplary techniquesalso include those disclosed in U.S. Pat. No. 4,440,859 issued 3 Apr.1984 to Rutter et al, U.S. Pat. No. 4,530,901 issued 23 Jul. 1985 toWeissman, U.S. Pat. No. 4,852,800 issued 15 Apr. 1986 to Crowl, U.S.Pat. No. 4,677,063 issued 30 Jun. 1987 to Mark et al, U.S. Pat. No.4,678,751 issued 7 Jul. 1987 to Goeddel, U.S. Pat. No. 4,704,362 issued3 Nov. 1987 to Itakura et al, U.S. Pat. No. 4,710,463 issued 1 Dec. 1987to Murray, U.S. Pat. No. 4,757,006 issued 12 Jul. 1988 to Toole, Jr. etal, U.S. Pat. No. 4,766,075 issued 23 Aug. 1988 to Goeddel et al andU.S. Pat. No. 4,810,648 issued 7 Mar. 1989 to Stalker.

In brief, the DNA is inserted into an expression vector, such as aplasmid, in proper orientation and correct reading frame for expression.If necessary, the DNA may be linked to the appropriate transcriptionaland translational regulatory control nucleotide sequences recognised bythe desired host, although such controls are generally available in theexpression vector. Thus, the DNA insert may be operatively linked to anappropriate promoter. Eukaryotic promoters include the CMV immediateearly promoter, the HSV thymidine kinase promoter, the early and lateSV40 promoters and the promoters of retroviral LTRs. Other suitablepromoters will be known to the skilled artisan. The expressionconstructs will desirably also contain sites for transcriptioninitiation and termination, and in the transcribed region, a ribosomebinding site for translation (e.g. see WO 98/16643).

The vector is then introduced into the host through standard techniques.Generally, not all of the hosts will be transformed by the vector and itwill therefore be necessary to select for transformed host cells. Oneselection technique involves incorporating into the expression vector aDNA sequence marker, with any necessary control elements, that codes fora selectable trait in the transformed cell. Such markers includedihydrofolate reductase, G418 or neomycin resistance for eukaryotic cellculture. Alternatively, the gene for such a selectable trait can be onanother vector, which is used to co-transform the desired host cell.

A typical mammalian cell vector plasmid is pSVL available from Pharmacia(Piscataway, N.J., USA). This vector uses the SV40 late promoter todrive expression of cloned genes, the highest level of expression beingfound in T antigen-producing cells, such as COS-1 cells. Examples of aninducible mammalian expression vectors include pMSG, also available fromPharmacia (Piscataway, N.J., USA), and pTet-off and pTRE2 available fromClontech (Catalogue Nos K1620-A and 6241-1, respectively, Clontech, PaloAlto, Calif., USA). The pMSG vector uses the glucocorticoid-induciblepromoter of the mouse mammary tumour virus long terminal repeat to driveexpression of the cloned gene. The pTet-off and pTRE2 vectors use thepresence or absence of tetracycline to induce protein expression via thetet-controlled transcriptional activator.

Host cells, for example murine Swiss 3T3 cells, that have beentransformed by the recombinant DNA encoding the transglutaminase arethen cultured for a sufficient time and under appropriate conditions topermit the expression of the transglutaminase and the subsequentcross-linking of ECM.

Thus, the kits of the invention may comprise a surface to whichcross-linked ECM material may be adhered and cells capable of expressinga tissue transglutaminase, either endogenously or as a result oftransfection (such as those identified above). Prior to using the kits,the cells are cultured in contact with the surface so as to depositcross-linked ECM material on said surface.

Alternatively, cells which naturally express low levels of a tissuetransglutaminase (or which do not naturally express a tissuetransglutaminase) may be cultured and an exogenous tissuetransglutaminase added in order to effect cross-linking of the ECM.Thus, in one embodiment, the kits of the invention may comprise asurface to which cross-linked ECM material may be adhered, cells whichnaturally express low levels of a tissue transglutaminase (or which donot naturally express a tissue transglutaminase), and an exogenoustissue transglutaminase. The exogenous tissue transglutaminase may bederived from natural sources (e.g. human tissue or cells such as lung,liver, spleen, kidney, heart muscle, skeletal muscle, eye lens,endothelial cells, erythrocytes, smooth muscle cells, bone andmacrophages) or may be produced by recombinant means (as describedabove).

It will be appreciated by persons skilled in the art that, in themethods and kits of the invention, the surface to which the cross-linkedECM material is adhered (or is to be adhered) may take one of a numberor forms. For example, the surface may be interior surface of a testtube or vial or the like. Alternatively, the surface may be concavesurface of a well in a single- or multi-well plate, for example amicrotitre plate.

Preferably, the surface is a multi-well plate such as a 96 well, flatbottomed, tissue culture treated plate (Product code M9780, SigmaAldrich Company Ltd, Fancy Road, Poole, Dorset BH12 4QH, UK).

In a preferred embodiment of the second aspect of the invention, the kitfurther comprises a negative control sample. Preferably, the negativecontrol sample is a serum sample or saliva sample from a healthyindividual. Conveniently, the control sample is lyophilised serum orsaliva.

Suitably, the control sample is an artificial or synthetic sample, suchas bovine serum albumin.

Conveniently, the kit further comprises a positive control samplecontaining antibodies which are capable of binding to cross-linked ECMand, hence, will provide a positive test result against which the sampleto be tested may be compared.

In an additional preferred embodiment of the second aspect of theinvention, the kit further comprises a secondary antibody. By ‘secondaryantibody’ we mean an antibody which can used to detect antibodies fromthe sera samples (i.e. primary antibodies) which have bound to thecross-linked ECM.

Preferably, the secondary antibody is an anti-human IgA antibody.

Alternatively, the secondary antibody is an anti-human IgG antibody.

In a preferred embodiment of the second aspect of the invention, the kitof parts is suitable for use in an ELISA.

Hence, the kit preferably comprises a secondary antibody which comprisesa detectable moiety such as a fluorescent label, radiolabel or enzyme.The detectable moiety may be linked to the secondary antibody directlyor via a linker moieties such as biotin-avidin.

Preferably, the secondary antibody is a goat anti-human IgA (α) antibodyconjugated to horse radish peroxidase (e.g. Cat. Number 31417ZZ, PierceChemical Co, Rockford, Ill., USA).

Advantageously, the kit further comprises a horse radish peroxidasesubstrate, for example o-phenylebediamine dihydrochloride inacid-buffered H₂O₂.

Preferably, the kit further comprises a stop reagent such as sulphuricacid to quench the reaction of the horse radish peroxidase with thehorse radish peroxidase substrate.

Conveniently, the kit further comprises a blocking agent such asdefatted milk powder to block non-specific sites in the cross-linked ECMmaterial.

Advantageously, the kit further comprises a washing agent such as adetergent to remove cellular material from the surface to which the ECMis adhered. For example, the washing agent may comprise a Tween® (e.g.Tween® 20), Triton-X100, CHAPS, CTAB or ammonium hydroxide solution.Preferably, the washing agent/detergent is dissolved inphosphate-buffered saline.

Preferred embodiments of the invention will now be described withreference to the following figures:

FIG. 1: ECM ELISA of human serum diluted 1/500 in which TGase inducedcells (minus tetracycline in medium) are compared with TG non-inducedcells (plus tetracycline in medium).

-   Key: CUB—anti-transglutaminase Type 2 murine monoclonal antibody,    CUB 7402 (Neomarkers), diluted 1/1000. FN—polyclonal rabbit    anti-fibronectin (Sigma F3648) at 1/1000.

FIG. 2: ECM ELISA of human serum diluted 1/500 showing the effect on theassay of using a variety of extractants to prepare the ECM.

-   Key: TX100—0.1% Triton X-100. CTAB—0.01% CTAB, CHAPS—0.01% CHAPS,    ammonium hydroxide—20 mM ammonium hydroxide in 0.1% Triton X-100.

FIG. 3: ELISA of ECM prepared from human dermal fibroblasts with humanserum diluted 1/500.

EXAMPLES Materials and Methods Preparation of Transfected Swiss 3T3Cells

In one embodiment, the ECM plate assay for the diagnosis of coeliacdisease is based on the use of murine Swiss 3T3 fibroblast cellstransfected with the gene for human type II transglutaminase (hTG) toproduce a cross linked extracellular matrix rich in hTG. The cells arecultured directly into the multiwell plates to be used in the assay sothat the ECM can be prepared as an antigen in an antibody capture typeELISA by the simple removal of the cells. This is made possible by theremoval of other cellular materials with a detergent extraction with0.1% sodium deoxycholate in phosphate buffered saline (or an equivalentbuffer) for 10 minutes at room temperature. Other detergents (TritonX-100, CHAPS, CTAB) and ammonium hydroxide (known to be efficient aspreparing ECM from cultured cells) have also been used with equalsuccess. The critical requirement here is that the modified ECM is leftintact on the bottom of the well while the remaining cellular materialsare removed.

Cell Lines

3T3 Swiss Albino mouse embryo fibroblasts were obtained from theEuropean Collection of Animal Cell Cultures (ECACC). They were culturedin Dulbecco's modified Eagle's medium (DMED4, Sigma-Aldrich Co Ltd,Poole, Dorset, UK) containing 10% fetal calf serum (FCS), 2 mMglutamine, 100 U/ml of penicillin and 100 μg/ml of streptomycin. Cellsexpressing the tetracycline-controlled transactivator (tTA) were kept inmedium supplemented with 400 μg/ml active G418 (Geneticin; GIBCO BRL,Life Technologies Ltd, Paisley, Scotland, UK). Stable cell linesoriginating from tTA and expressing tissue transglutaminase werecultured in medium further supplemented with 250 μg/ml xanthine(Sigma-Aldrich Co Ltd), 15 μg/ml hypoxanthine, 10 μg/ml thymidine, 2μg/ml aminopterin, and 10 μg/ml mycophenolic acid (GIBCO-BRL, LifeTechnologies Ltd).

Plasmid Vectors

The expression plasmid pUHD15-1 encoding tTA under the control of theHCMV promoter/enhancer, pUHC13-3 encoding luciferase under the controlof the tTA-dependent minimal promoter hCMV*-1 and the tTA-dependentexpression vector pUHD10-3 containing PhCMV*-1, followed by a multiplecloning site, were generously provided by H Bujard (Zentrum furMolekulare Biologie, Heidelberg, Germany).

The plasmid pUHD10-3-TG was constructed by inserting the full lengthhuman tissue transglutaminase cDNA (Gentile et al, supra) into the EcoRIsite of pUHD10-3.

The neomycin resistance plasmid pSVneo and the expression vectorpSNFβGal encoding β-galactosidase were a gift of P. J. A. Davies(University of Texas Health Centre at Houston). The plasmid pUS1000encoding XGPT (xanthine-guanine phosphoribosyltransferase) was donatedby P Sanders (University of Surrey).

Transfections

Swiss 3T3 cell lines stably expressing the tetracycline-controlledtransactivator (tTA clones) were generated using liposome-mediatedtransfection as previously described in Johnson et al, 1994, Oncogene9:2935-2942. Cells (1.0×10⁶) were seeded into a 10-cm tissue culturepetri dish 1 day prior to DNA transfer and then cotransfected with 9 μgof pUHD15-1 and 1 μg of pSVneo, using 100 μg of lipofectin per 10 μg ofDNA (GIBCO-BRL, Life Technologies Ltd). Clones resistant to 800 μg/ml ofactive G418 (Geneticin, GIBCO-BRL, Life Technologies Ltd) were screenedfor tTA activity by transient transfection of pUHC13-3 using calciumphosphate (Kingston, 1990, In: Current Protocols in Molecular Biology,Ausubel et al. (Eds.), Greene-Wiley Interscience, New York) and thenscreened for luciferase activity (luciferase assay system, Promega Ltd,Southampton, UK). Light intensity was evaluated utilizing ascintillation counter and expressed in cpm per micrograms of protein.Transfection efficiency was normalized by cotransfection with 0.5 μg ofPSVβgal and measurement of β-galactosidase activity (β-galactosidaseassay system, Promega Ltd).

Positive clones were selected for further cotransfection withpUHD10-3-TG (9 μg) and the XGPT expression plasmid pUS1000 (μg) usinglipofectin. Tetracycline (1 μg/ml) was included in the culture medium.Transfected clones resistant to selection medium for the salvage enzymeXGPT (250 μg/ml xanthine, 15 μm/ml hypoxanthine, 10 μg/ml thymidine, 2μ/ml aminopterin, 10 μg.ml mycophenolic acid) were analysed for theircapability to overexpress tissue transglutaminase (tTGase) byimmunocytochemical staining, following withdrawal of tetracycline for 72h. Clones were also screened for expression of tTGase by immunoprobingof Western blots of lysates as described previously (Johnson et al,supra). Western blots were probed using the monoclonal antibody toguinea pig liver enzyme Cub 7402 diluted 1/3000 in 0.1 M Tris-HCl, pH7.4 (Birckbichler et al, 1985, Hybridoma 4:179-186) and revealed bychemiluminescence (Amersham International plc, Little Chalfont, Bucks,UK) using an anti-mouse IgG HRP conjugate. Guinea pig livertransglutaminase (Sigma-Aldrich Co. Ltd) was used as a tTGase standard.Protein concentration was determined using the method of Lowry.

Induction of Transglutaminase Expression in Transfected Cells

Cell lines stably transfected with transglutaminase containingpUHD10-3-TG were continuously cultured in the presence of tetracycline 2μg/ml. To induce transglutaminase expression, exponentially growingcells were cultured in the absence of tetracycline for a minimum of 72 hand a maximum of 96 h. Parallel control cultures grown identically,keeping tetracycline in the medium. During the period of induction,culture medium was changed every 48 h.

Analysis of Transglutaminase Activity

Transglutaminase activity was measured as described in Lorand et al,1972, Anal. Biochem. 50:623-631.

Cell Culture and Antigen Preparation

Culture of the transfected Swiss 3T3 cell line has been optimised tomaximise the deposition of ECM in a form favourable to the endpoint ofthe assay. We have shown that the antigenic nature of the ECM preparedfrom these cells is optimal if one allows for complete induction of hTGin the first instance and for maturation of the ECM into a more ‘tissuelike’ structure in the second instance. Immunofluorescent staining ofcultured transfected Swiss 3T3 cells with coeliac sera has shown thatthe ECM antigen is most dense where the cells are confluent suggestingthat antigen production is maximised during cell contact. We have foundthat cells cultured for 1 day past confluence produce ECM that is mostsuited for the assay.

Growing stocks of the transfected Swiss 3T3 cells are maintained inmedia designed to suppress expression of the hTG and to maintain theclonal identity of the cell line. The formula for this media is givenbelow.

For every 500 ml of DMEM (Sigma 5546) remove 100 ml of media and add:

-   -   b) 50 ml foetal calf serum;    -   c) 10 ml 200 mM L-glutamine solution;    -   d) 10 ml 50× penicillin/streptomycin solution (10,000 units        penicillin/10 mg streptomycin/ml);    -   e) 10 ml 50×HAT supplement (Sigma H8016);    -   f) 10 ml xanthine stock. Stock prepared by dissolving 250 mg of        xanthine in 4 ml of 1M NaOH and adding 16 ml dH₂O;    -   g) 10 ml mycophenolic acid stock (prepared by dissolving 10 mg        mycophenolic acid in 2 ml 1M NaOH and adding 18 ml dH₂O);    -   h) 2 ml G418 stock (prepared from powder at 1 g/10 ml in        distilled water) and    -   i) 0.1% v/v tetracycline in distilled water.

As the tetracycline degrades in solution within 2 or 3 days, it isimportant to make up only enough complete media for each batch ofculture. To maintain suppression of the hTG in culture the media shouldbe replaced every other day. Induction of the hTG gene is simplyachieved by replacing the culture media with similar fresh media free ofthe suppressor tetracycline. For the purposes of this example, theinduced cells were left for 4 days (refreshing the media after 2 days)to ensure complete hTG induction. After the induction period, cells weretransferred to the culture plates that were used in the assay, usually96 well at a seeding density of 9000 cells per well and left incubatedfor a further 3 days. As these cells are adherent, it was necessary touse a standard trypsinisation step to remove the cells from the flaskwall to enable sub-culturing and propagation.

To prepare the antigenic ECM, the culture medium must first be removedand the wells washed gently in phosphate buffered saline pH 7.4 (PBS).Unwanted cellular material is then removed by incubating each well with100 μl of 0.1% sodium deoxycholate in PBS for 10 minutes. The wellcontents are then discarded and washed twice with PBS 0.2% Tween®-20(PBST) followed by one wash with PBS. Non-specific binding sites arethen blocked with 100 μl/well of 3% defatted milk powder (Marvel) in PBS(PBSM) for 30 minutes at 37° C. Plates can be stored at −20° C. in thisstate for at least one month. Longer storage may be possible.

The actual assay follows a basic protocol for an antibody capture assayas detailed below. It is envisaged that each kit may include negativeand positive control sera (or saliva) consisting of screened human sera(or saliva). The negative control sample could be pooled from a group ofpositively identified non-coeliacs and the positive control sample wouldmost likely consist of one high and one low titre coeliac serum orsaliva. Both may be pre-diluted to the working concentration ifpossible. All samples and standards are to be assayed in triplicate.

-   -   1. Discard the blocking solution and add 50 μl per well of human        serum to be tested diluted 1/500 in PBSM. Incubate for 2 hours        at 37° C.    -   2. Wash twice with PBST and once with PBS.    -   3. Add 50 μl per well of anti-human IgA conjugated to HRP        diluted appropriately in PBSM. For example, Pierce goat        anti-human IgA (α chain specific) at 1/5000 in PBSM. Incubate        for 2 hours at 37° C.    -   4. Wash twice with PBST and once with PBS.    -   5. Make up fresh HRP substrate based on o-phenylenediamine        dihydrochloride (OPD) in acidified buffered H₂O₂. For example,        one tablet of OPD (Sigma P3804, 5 mg/tablet) and 2 tablets of        phosphate-citrate buffered urea H₂O₂ (Sigma P4560) to 20 ml        dH₂O.    -   6. Plate out substrate solution at 50 μl per well and leave for        30 minutes at room temperature. Stop reaction with 50 μl per        well of 2.5M H₂SO4₄ and read plate at 492 nm.

Saliva samples may be assayed in a similar manner.

Example 1

1. Sera Used

FIRST BATCH: Obtained from Iain Murray, then of the City Hospital,Nottingham.

Patient Brief Pathology/Diagnosis 12 Untreated CD 13 Diarrhoea 14Untreated CD (EMA+) 15 Diarrhoea, not CD 18 Normal 27 Normal 53Untreated CD

SECOND BATCH: Obtained from Iain Murray, then of Barnsley GeneralHospital.

Patient Brief Pathology/Diagnosis 2 CD with total villus atrophy 3Normal 4 CD showing mild villus blunting, increased intra-epitheliallymphocytes and increased anti-gliadin antibodies 9 CD on a gluten freediet showing focal mild villus atrophy 12a Abnormal gut morphology butnot indicative of CD 13a CD showing increased intra-epithehallymphocytes and partial villus atrophy 14a CD showing total villusatrophy 17  CD showing total villus atrophy

Supplemented by two samples from Nottingham Trent Universitypost-graduate students, designated C1 and C2.

2. Method

As described above.

3. Results

FIG. 1 shows data from an ECM ELISA of human serum diluted 1/500. TGinduced cells (minus tetracycline in medium) are compared with TGnon-induced cells (plus tetracycline in medium).

The results shown in FIG. 1 are typical of those obtained with ECMderived from Swiss 3T3 fibroblasts. All sera from patients diagnosed bybiopsy as suffering from coeliac disease give large absorbance valueswhen compared with the controls and known non-coeliac sera. Note that anindirect ELISA of these sera against human serum albumin, i.e. anunrelated antigen, gave signals similar to those found with thecontrols. Thus, the signals obtained with the controls and non-coeliacsera can be considered to represent the level of background for thisassay. For example, it is possible to determine positive signals simplyas those that are at least twice the average background.

An observable difference between induced type II TG and non-induced typeII TG was limited to patients 4 and 53 and was only 10-20% of thesignal, suggesting that antigenic TG is not the most significant antigenin this assay. Table 1 shows the results of other indirect ELISAs withguinea pig liver TG or human fibronectin as antigens compared with ECMand a control of human serum albumin for some of the sera.Anti-fibronectin activity of the sera was looked at as this protein isrecognised as a principal constituent of the ECM and we hypothesisedthat anti-fibronectin antibodies were likely components in a complexpolyclonal anti-ECM antibody profile. Even with this small sample ofsera this simple comparison has illustrated deficiencies in the singleantigen approach. Patients 14 and 17 were negative in the gpTG assay butwere positive in the ECM assay and patients 4 and 9 were negative in thefibronectin assay but were positive in the ECM assay. It is particularlyinteresting to note that patient 4 is assayed as negative with gpTG andfibronectin but is positive (in accordance with the clinical diagnosis)in the ECM based assay. It was also noted that the fibronectin basedassay was prone to weak false positives with two patients, numbers 3 and12 giving incorrect results.

TABLE 1 EMC ELISA compared to simple indirect ELISAs human serum albumin(HSA), guinea pig liver transglutaminase (gpTG), and human fibronectin.TVA—total villus atrophy, PVA—partial villus atrophy. Patient HSA gpTGFN ECM- Pathology  2 − +++ ++ +++ TVA  3 (+) − + − Normal  4 − − − + PVA 9 − ++ − ++ PVA 12 − − + − Abnormal 13 − ++ ++ +++ PVA 14 − − + ++ TVA17 ++ (+) +++ +++ TVA 25 − − − − Normal C1 − − − − Control 1 C2 − − − −Control 2 Key: − Background, (+) Weak positive, + Low Positive, ++Medium Positive, +++ High Positive.

Based on these data we are proposing that the improvement in the cellmatrix assay over existing assays comes from the antigen being more likethe material that may be targeted by the immune system during theprogression of coeliac disease. That is to say that although type II TGhas been shown to be an antigen in most cases of coeliac disease ourresults support the findings of other researchers that have shown thatTG is not the only antigen and that this may be exploited to produce abroader based assay.

Example 2

1. Sera Used

See Example 1.

2. Method

This method is essentially the same as that given previously except that0.1% Triton X-100, 0.01% CTAB, 0.1% CHAPS and 20 mM ammonium hydroxidewith 0.1% Triton X-100 replace the 0.1% sodium deoxycholate.

3. Results

FIG. 2 shows data from an ECM ELISA of human serum diluted 1/500 to showthe effect on the assay of using a variety of extractants to prepare theECM.

The data in FIG. 2 show that the endpoint of the assay is essentiallyindependent of the method used to prepare the matrix proteins for use inthe assay. For the assay to work, the extraction needs to remove thegeneral bulk of the cellular material and leave matrix proteins behindin a way that preserves the antigenic qualities of this material. Thevariation that is evident in FIG. 2 can be attributed to naturalfluctuations in the character of the cell matrix as it is laid down andto general inter-assay variations that occur in all ELISAs before propernormalisation has been applied. Even so, this amount of variation in theendpoint does not prove to be a problem when identifying positive sera.

Example 3

1. Sera Used

Second batch of sera used in Example 1.

2. Method

Screened primary cultures of human foreskin dermal fibroblasts(preferably not older than passage 10) were maintained in complete DMEM(Sigma 5546) containing 10% FCS, 2% 200 mM L-glutamine solution and 2%penicillin/streptomycin solution (10,000 units penicillin, 10 mgstreptomycin/ml) using standard tissue culture methods. Cells weretransferred to the culture plates that were used in the assay, usually96 well, at a seeding density of 10000 cells per well and left toincubated for a further 3 days. As these cells are adherent, it isnecessary to use a standard trypsinisation step to remove the cells fromthe flask wall to enable subculturing and propagation.

3. Results

FIG. 3 shows data from the ELISA of ECM prepared from human dermalfibroblasts with human serum diluted 1/500.

On the whole, the results with the human foreskin dermal fibroblastgenerated ECM mirrored those seen with the 3T3 derived ECM. However, ascan be seen in FIG. 3, the signal is diminished to the point wherepatient 4 is not distinguishable from the control.

1. A method for diagnosing coeliac disease in a patient comprising: (a)contacting an antibody sample from said patient with insolublecross-linked extracellular matrix (ECM) material immobilised on asurface, wherein the ECM material is derived from cells which express atissue transglutaminase and comprises proteins selected from the groupconsisting of fibronectin, vitronectin, collagens, latent TGFβ-1 bindingprotein (LTBP-1), fibrillins, elastin and laminin, wherein said ECM istreated prior to use to remove non-ECM cellular material; and (b)detecting binding of two or more types of antibodies present in thesample to the immobilised ECM material, each type of antibody havingaffinity for a different protein component of the cross-linked ECM,wherein the binding of antibodies present in the sample to theimmobilised ECM material is indicative of a positive diagnosis.
 2. Amethod according to claim 1 wherein the antibody sample is a serumsample.
 3. A method according to claim 1 wherein step (b) comprisesdetecting binding of IgA antibodies to the immobilised ECM material. 4.A method according to claim 1 further comprising repeating steps (a) and(b) with a negative control sample.
 5. A method according to claim 4,wherein the negative control sample is a serum sample or saliva samplefrom a healthy individual.
 6. A method according to claim 4 wherein apositive diagnosis is made when the level of binding of antibodiespresent in the patient's antibody sample to the immobilised ECM materialis at least 20% greater than the level of binding of antibodies presentin the negative control sample.
 7. A method according to claim 6 whereina positive diagnosis is made when the level of binding of antibodiespresent in the patient's antibody sample to the immobilised ECM materialis at least 100% greater than the level of binding of antibodies presentin the negative control sample.
 8. A method according to claim 6,wherein a positive diagnosis is made when the level of binding ofantibodies present in the patient's antibody sample to the immobilisedECM material is at least 200% greater than the level of binding ofantibodies present in the negative control sample.
 9. A method accordingto claim 1 wherein step (b) comprises using an ELISA.
 10. A methodaccording to claim 9 wherein the ELISA comprises using an anti-human IgAantibody.
 11. A method according to claim 1 wherein the cells aretransfected with a nucleic acid molecule encoding a tissue (type II)transglutaminase.
 12. A method according to claim 11 wherein the cellsare Swiss 3T3 cells.
 13. A method according to claim 1 wherein thetissue transglutaminase is human type II transglutaminase.
 14. A methodaccording to claim 1 wherein the surface is a multi-well plate.
 15. Amethod according to claim 1, wherein the ECM material is derived fromcells transfected with a nucleic acid molecule encoding atransglutaminase.